Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

GFP variants are highly stable to proteasomal degradation, so GFP-linked fragments are commonly observed to accumulate in cells. 54Movie of split talin photoactivation in live cells.Talin knock-out cells were transfected with LifeAct-mNeonGreen, Talin head-SpyTag003, SpyCatcher003(K31HCK)-Talin rod-mScarletI and HCK RS and cultured with 0.25 mM HCK for 16-18 h.Left column shows inverted signal of actin labeling with LifeAct-mNeonGreen, middle column shows bright-field, and right column shows a merge of bright-field (grayscale) with LifeAct-mNeonGreen (green) determined by confocal microscopy.Top row: at 1 min 50 s, a circular 4-µm region was photoactivated by 405 nm illumination for 5 s, indicated by a magenta circle.Bottom row: cells treated in the same way but without 405 nm illumination.Photoactivation triggers initial local extension of the lamellipodium, followed by global cell spreading as reconstituted talin diffuses throughout the cell.The scale bar is 10 µm and the movie is shown at 6 frames per s (30-fold sped-up).
To avoid possible phototoxicity of reactive oxygen species in the 405 nm activation of HCK in living cells, 0.25 mM Trolox (Sigma-Aldrich) was added as an antioxidant. 50100 mM stock of Trolox was prepared in 99.7 % (v/v) ethanol and stored at 4 °C.

Expression of caged SpyCatcher003 in HEK293T cells
HEK293T cells were maintained in complete Dulbecco's Modified Eagle Medium [DMEM (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), as well as 50 U/mL penicillin and 50 µg/mL streptomycin (Thermo Fisher)] under humidified conditions at 37 °C and 5% (v/v) CO2.The day before transfection, 250,000 HEK293T cells were seeded in 2 mL complete DMEM in 6-well plates (Greiner).Cells were transfected with 2 μg plasmid DNA (1 part pcDNA3-EGFP-Talin head-SpyCatcher003 WT/K31TAG and 2 parts pE323-HCK RS) using 2 μL jetOPTIMUS (Polyplus) according to the manufacturer's instructions.HCK was added to a final concentration of 0.25 mM after transfection.For control wells, 0.25% (v/v) DMSO was added.Cells were grown for 48 h, before Western blot analysis with the plates wrapped in aluminum foil.
For immunoblotting in Fig. 1E, Talin knock-out cells cultured on 60 mm dishes were briefly washed with PBS and lysed with 150 µL of 5× SDS-PAGE sample buffer: 250 mM Tris-HCl, pH 6.8, 10% (w/v) SDS, 30% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 25 mM tris(2-carboxyethyl)phosphine (TCEP).Lysed cells were collected to the dish corner with cell scrapers and the lysate was transferred to 1.5 mL tubes cooled on ice.Samples were heated for 5 min at 95 °C, briefly spun down to collect liquid, and stored at -20 °C before blotting.

Initial optimization of caged SpyCatcher003 expression in HEK293T cells
Optimization was performed in 24-well plates (Greiner).HEK293T cells were seeded at 50,000 cells/mL in 0.5 mL complete DMEM.Cells were transfected with 0.25 μg total DNA using 0.25 μL jetOPTIMUS, at molar ratios 1:1, 1:2 and 2:1 [pcDNA3-TfR-SpyCatcher003(K31TAG)-sfGFP to pE323-HCK RS].Directly after transfection, HCK was added to a final concentration of 0.25 mM, 0.5 mM or 1 mM.Western blot analysis was performed 48 h after transfection.For lysis, HEK293T cells were washed three times with 0.5 mL PBS, then incubated in 75 μL ice-cold lysis buffer and processed as described above.

Protein purification by Ni-NTA
SpyTag003-MBP was expressed in E. coli EXPRESS BL21(DE3) (Lucigen) and purified by Ni-NTA at 4 °C.Bacterial cell pellets of SpyTag003-MBP were resuspended in Ni-NTA buffer (50 mM Tris-HCl pH 7.8 + 300 mM NaCl) supplemented with cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche) and 1 mM PMSF.Cells were lysed by sonication on ice at 50% duty cycle for 4 × 1 min with 1 min rest between runs.Cell lysates were clarified by centrifugation at 30,000 g for 30 min at 4 °C, then incubated with Ni-NTA agarose (Qiagen) for 1 h at 4 °C with rolling.Resin was pelleted by centrifugation, then washed with 20 column volumes (CV) of Ni-NTA buffer and Ni-NTA wash buffer (50 mM Tris-HCl + 300 mM NaCl + 10 mM imidazole at pH 7.8).The resin was transferred to Econo-Pac Chromatography Columns (Bio-Rad) and washed with a further 20 CV of Ni-NTA wash buffer by gravity flow.Elution was performed with Ni-NTA elution buffer (50 mM Tris-HCl + 300 mM NaCl + 200 mM imidazole at pH 7.8) by incubating the resin with 4 × 1 CV for 5 min.SpyTag003-MBP was dialyzed into PBS in three dialysis steps using 3.5 kDa molecular weight cut-off (MWCO) dialysis tubing (Spectrum Labs).Protein concentrations were determined by A280 measurement on a NanoDrop One (Thermo Fisher) using NanoDrop One software version 1.4.2, with extinction coefficients predicted by ExPASy ProtParam. 52tracellular 405 nm uncaging for Western blot Talin knock-out cells were transfected with pcDNA3-EGFP-Talin head-SpyCatcher003(K31TAG), pEGFP-C1 SpyTag003-Talin rod (434-2541)-mCherry and HCK RS plasmids as described below, and plated on a 60 mm dish at 2 × 10 6 cells per dish.After an initial 2 h recovery phase at 37 °C and 5% (v/v) CO2, FluoroBrite DMEM was replaced with FluoroBrite DMEM supplemented either with 0.25 mM HCK (100 mM stock in DMSO) and 0.25 mM Trolox or with 0.25 % DMSO and 0.25 mM Trolox.Incubation at 37 °C and 5% (v/v) CO2 was continued for 17 h, after which cells were washed twice with FluoroBrite DMEM supplemented with 0.25 mM Trolox and incubated at 37 °C and 5% (v/v) CO2 for 15 min.For intracellular SpyCatcher003(HCK) uncaging, culture dishes were placed for 1 min on 2 × 6 W 405 nm LED panels (Sovol) with total intensity of 45 mW/cm 2 at the plane of cells.Activated cells were allowed to recover at 37 °C and 5% (v/v) CO2 for 5 min and the activation cycle was repeated two more times for a total activation time of 3 min.Cells were incubated at 37 °C and 5% (v/v) CO2 for 1h 30 min and lysed for Western blot as described below.

Uncaging and reaction of SpyCatcher003 in cell lysates
Equal amounts of protein lysates as determined by BCA assay were transferred to PCR tubes.To test spontaneous uncaging by ambient light, samples were incubated exposed to room light in a cold room or next to a window on ice for the indicated amounts of time.For uncaging, designated samples were irradiated on a ChemiDoc XRS+ using the Ethidium Bromide setting for the indicated amounts of time at 25 °C.Samples were transferred back to ice to chill and incubated with 50 μM SpyTag003-MBP at 4 °C for 1 h.Reaction was stopped by mixing with 6× SDS loading buffer [234 mM Tris-HCl pH 6.8, 24% (v/v) glycerol, 120 μM bromophenol blue, 234 mM SDS, supplemented with 60 mM dithiothreitol (DTT) for reduced samples] and heating at 95 °C for 5 min in a Bio-Rad C1000 thermal cycler.
Western blot SDS-PAGE in Fig. 1E was performed using Bio-Rad Mini-PROTEAN Tetra Vertical Electrophoresis Cell with Bio-Rad 4-15% Mini-PROTEAN TGX Precast gels.Gels were run with in 24 mM Tris base, 192 mM glycine, 3.5 mM SDS at 120 V. Proteins were transferred to nitrocellulose membranes using Bio-Rad Trans-Blot Turbo Transfer System at 1.3 A and 25 V for 10 min.Membranes were blocked with Bio-Rad EveryBlot blocking solution for 1 h 30 min at 25 °C.Anti-EGFP antibody (SICGEN ab0020) was diluted 1:1,000 and anti-mScarlet (also recognizing mCherry) antibody (SICGEN ab9088) was diluted 1:2,000 directly into the blocking solution.Primary antibodies were incubated for 16 h at 4 °C with gentle agitation.Membranes were washed four times in 0.05% TBST [50 mM Tris-HCl, 150 mM NaCl, pH 7.4 + 0.05% (v/v) Tween 20] for 5 min at 25 °C.Secondary antibody detection was performed by staining for 1 h 30 min at 25 °C with Licor IRDye 680RD donkey anti-goat IgG (Licor 926-68074) diluted 1:20,000 into Bio-Rad EveryBlot blocking buffer.Membranes were washed five times in 0.05% TBST before a final wash with TBS.Membranes were imaged using Licor CLx imager with 84 µm resolution and medium image quality.SDS-PAGE in Fig. S1A-C and Fig. S2 was performed using an XCell SureLock system (Thermo Fisher) with 10% (w/v) polyacrylamide gels.Gels were run in 24 mM Tris base, 192 mM glycine, 3.5 mM SDS at 200 V. Proteins were transferred onto nitrocellulose membranes by dry transfer using an iBlot 2 gel transfer device (Thermo Fisher) at 25 V for 10 min.Membranes were blocked for 1 h at 25 °C in 5% (w/v) skimmed milk in PBS.The primary antibody was diluted in 2.5% (w/v) skimmed milk in 0.05% PBST [PBS + 0.05% (v/v) Tween 20], with mouse anti-GFP (Thermo Fisher, MA5-15256, clone GF28R) at 1:2,000 or mouse anti-SpyCatcher003 serum 53 at 1:300.Incubation was performed overnight at 4 °C.Membranes were washed three times in 0.1% PBST [PBS + 0.1% (v/v) Tween 20] for 5 min at 25 °C.Secondary antibody detection was performed by staining with goat anti-mouse IgG Horseradish Peroxidase (Sigma-Aldrich, A4416) at 1:5,000 for 1 h at 25 °C in 2.5% (w/v) skimmed milk in 0.05% PBST.After three further washes in 0.1% PBST and one wash in PBS at 25 °C, membranes were developed at 25 °C with SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).In immunoblotting with anti-EGFP antibody, we observed EGFP-containing degradation products of EGFP-Talin head-SpyCatcher003(K31TAG), consistent with reported high stability of EGFP in cells. 54A colon is used to indicate products linked by Tag/Catcher isopeptide bond formation.

Phototoxicity assay for 405 nm wide-field activation
Talin knock-out cells were plated on Nunclon Delta-Treated 96-well plates (Nunc) at 50,000 cells per well.Cells were incubated at 37 °C and 5% (v/v) CO2 for 1 h in 100 µL FluoroBrite DMEM (Thermo Fisher) supplemented with either 0.25 mM Trolox (Sigma-Aldrich, 100 mM stock in ethanol) or matching ethanol concentration at 0.25 % (v/v).Cells were exposed to 405 nm light (45 mW/cm 2 ) from a wide-field LED light source for 0.5-10 min and allowed to recover for 15 min at 37 °C and 5% (v/v) CO2.20 µL resazurin-based CellTiter-Blue reagent (Promega) was added per well, before the plates were gently mixed and incubated for 4 h at 37 °C and 5% CO2.Plates were cooled to 21 °C and 80 µL samples of culture media were transferred to black half-area microplates (Nunc).Media fluorescence at 550 nm excitation wavelength and 590 nm emission wavelength was measured using a Tecan Spark microplate reader.The linearity of CellTiter-Blue fluorescence signal at the used cell density and incubation time was confirmed with control experiments.Media-only controls with FluoroBrite DMEM supplemented with either 0.25 mM Trolox or 0.25% (v/v) ethanol and exposed for 0-10 min at 405 nm were used to correct results for changes in media fluorescence.All results were normalized by the fluorescence of unexposed cells cultured in matching media conditions.

Live-cell imaging and on-stage 405 nm photoactivation
Zeiss high-performance 170 µm-thick coverslips were washed with 2% (v/v) Hellmanex-III (Sigma-Aldrich) in a bath sonicator at 40 °C for 20 min (Finnsonic), rinsed with deionized water, and sonicated again in 96% (v/v) ethanol.Coverslips were rinsed with deionized water, air-dried, and attached to perforated 35 mm polystyrene dishes (MatTek).Attached coverslips were coated with 25 µg/mL human fibronectin in PBS for 30 min at 37 °C and washed twice with PBS.Fibronectin was purified from human plasma preparate (Octaplas) using gelatin affinity chromatography (Gelatin Sepharose 4B; GE Healthcare) as previously described. 55ransfected talin knock-out cells were plated on glass bottom dishes (166,000 cells per dish) in 120 µL complete FluoroBrite medium and were incubated at 37 °C and 5% (v/v) CO2 for 1 h to allow cell attachment.Dishes were filled with complete FluoroBrite medium and the incubation at 37 °C and 5% (v/v) CO2 continued for 2 h.Media was replaced with 800 µL of FluoroBrite-Trolox-HCK (complete FluoroBrite medium supplemented with 0.25 mM Trolox and 0.25 mM HCK) and cells were incubated at 37 °C and 5% (v/v) CO2 for 16-18 h.Before imaging, media was replaced with FluoroBrite-Trolox (complete FluoroBrite medium supplemented with 0.25 mM Trolox) and the dish was mounted to a humified 37 °C and 5% (v/v) CO2 incubator on the microscope stage.
For live-cell imaging and on-stage 405 nm photoactivation, we used a Nikon Eclipse Ti2-E inverted microscope equipped with A1R+ laser scanning confocal (Nikon), Perfect Focus System (Nikon) and Nikon SR HP Plan Apo 100×/1.35Silicone immersion objective.Single cells were imaged at 80 nm pixel size and 67.7 µm pinhole size using 488 nm solid state laser (0.25 -0.5% laser) for mNeonGreen excitation and 561 nm solid state laser (0.2 -0.5% laser) for mScarletI excitation.We used 560 nm long-pass dichroic mirror and 525/50 nm band-pass filter for mNeonGreen emission and 640 nm long-pass dichroic mirror and 595/50 nm band-bass filter for mScarletI emission.
Differential Interference Contrast (DIC) images were captured using transmitted 488 nm excitation light.Images were captured at 5 s intervals for 23 images before photoactivation.For HCK photoactivation, a circular region of 4.0 µm was activated for 5 loops (4.8 s total) with 10.5 µW (5%) 405 nm laser.Imaging was then immediately continued for 73 images at 5 s intervals.
"Unactivated" cells were cultured with HCK but not 405 nm activated.In Fig. 3, "No HCK" cells were cultured without HCK, activated with 405 nm, and allowed to spread for 90 min.When green and magenta images are merged, the overlapping regions appear white.

Lamellipodium extension analysis
Fiji distribution of ImageJ 1.53t 56 was used to analyze lamellipodium extension after HCK photoactivation.A 10-pixel (0.8 µm) wide line perpendicular to the cell edge was drawn across the cell lamellipodium at the site of photoactivation.A kymograph of mNeonGreen intensity was created along this line, covering all 96 frames of the time-series.Using free-hand selection tool, the movement of the lamellipodium edge over time was traced in the kymograph by following the shift in the position of mNeonGreen intensity.The selection created in this way was used to quantify the number of pixels above the line (inside cell) and below the line (outside cell) for all columns of pixels, each representing a single image captured at 5 s intervals.For relative lamellipodium extension, the results were normalized with the initial position of the lamellipodium edge.To calculate the lamellipodium extension rate, sharp irregularities in the extension data were smoothed by averaging with a 4-frame sliding window and the rate of change over time was calculated.

Actin flow-rate analysis
To improve the clarity of LifeAct-mNeonGreen live-cell imaging data, Huygens Essential 23.04 (Scientific Volume Imaging) with classic maximal likelihood estimation (CMLE) deconvolution algorithm was used.Deconvoluted images were analyzed using Fiji distribution of ImageJ 1.53t. 56Briefly, at the site of photoactivation, a 20-pixel (1.6 µm) wide line perpendicular to the cell edge was drawn across the lamellipodium to create 5 parallel kymographs, each representing the mean intensity of 4 pixels in the original image.These kymographs were used to measure manually the slope of actin flow at 30 s intervals (6-pixel spacing in the kymographs) using the line selection tool.Of the 5 parallel kymographs, at each time-point, the one with the best image clarity was selected for analysis.The slopes measured in this way were used to calculate actin flow rate (nm/s) using trigonometry.

Widefield 405 nm activation of talin knock-out cells
Transfected talin knock-out cells were plated on 60 mm dishes (Nunc) in complete FluoroBrite medium and allowed to recover for 3 h at 37 °C and 5% (v/v) CO2.The media was replaced with FluoroBrite-Trolox-HCK and the incubation at 37 °C and 5% (v/v) CO2 was continued for 16-18 h.After the addition of FluoroBrite-Trolox-HCK, all subsequent culture steps were performed in low light conditions.Cells were rinsed with PBS, detached using TrypLE Express (Thermo Fisher) and plated in FluoroBrite-Trolox-HCK on fibronectin coated glass-bottom dishes, prepared as described above.After 1 h incubation at 37 °C and 5% (v/v) CO2, cells were rinsed once with FluoroBrite-Trolox and incubated at 37 °C and 5% (v/v) CO2 for 15 min to wash out residual HCK.For HCK uncaging, dishes were placed on top of 2 × 6 W 405 nm LED panels (Sovol) with 45 mW/cm 2 total intensity and photoactivated for 60 s.Photoactivated cells were incubated at 37 °C and 5% (v/v) CO2 and fixed at defined timepoints.For fixation, media was aspirated and 4% (w/v) paraformaldehyde in PBS was added for 20 min at 22 °C.Cells were washed 4 times with PBS and stored at 4 °C.

Cell size and morphology analysis
For cell size and morphology analysis, mScarletI signal of fixed samples was imaged using a Nikon Eclipse Ti2-E inverted microscope equipped with A1R+ laser scanning confocal unit (Nikon) and Nikon Apo 60×/1.40Oil λS DIC N2 objective.For No HCK samples (Fig. 3B,C) not expressing mScarletI because of polypeptide chain termination, anti-vinculin antibody staining was imaged instead.
Cell area and circularity were analyzed in Fiji distribution of ImageJ 1.53t 56 using the free-hand selection tool to trace cell boundaries.Cell circularity was calculated with the formula: circularity = 4π(area/perimeter 2 ).Cells with partially detached lamellipodium or extremely high mScarletI signal were excluded from the analysis.For each timepoint, 45 to 110 cells from 2 independent experiments were analyzed.

Adhesion intensity analysis
For adhesion intensity analyses, immunostained cells were imaged using a Zeiss Axio Observer.Z1 equipped with Zeiss LSM800 laser scanning confocal unit and Zeiss Plan-Apochromat 63×/1.40 oil immersion objective.107 µm × 107 µm fields were imaged at 70 nm pixel size and 200 nm Z-stack interval, maintaining fixed laser intensities and detector gains for all samples.
Adhesion protein localization to adhesion sites was analyzed using Fiji distribution of ImageJ 1.53t. 56The Z-stack slices with the highest adhesion/cytosol contrast were selected for the analysis.For each cell, the 5 adhesions with the highest fluorescence intensity were masked using the freehand selection tool and the mean intensity of each selected region was measured.For each image, the intensity of background control region outside the cell was measured and its intensity value was subtracted from all adhesion intensity values of the same image.For Fig. 3F, adhesion intensity values were normalized by the mean intensity measured at the 90 min timepoint.For unactivated cells, no clear adhesion structures were observed and the reported intensity values represent mean lamellipodium intensity.For each adhesion protein, 60-85 adhesions in 12-17 cells from two independent experiments were analyzed.Statistical significance of results was tested using one-way ANOVA with Dunnett's post-test in GraphPad Prism 9.0.0 (GraphPad Software).All other samples were compared with the unactivated samples.p-values below 0.05 were considered statistically significant.p-values above 0.05 were considered non-significant (ns).Samples in the same experiment were prepared, imaged and analyzed under uniform conditions.

AFigure S1 .
Figure S1.Validation of SpyCatcher003(K31HCK) photoactivation.(A) Titration of HCK concentration and plasmid ratios.HEK293T cells were transfected with different molar ratios of TfR-SpyCatcher003(K31TAG)-sfGFP and HCK RS plasmids and cultured in the presence of 0.25 mM -1.0 mM HCK. Cell lysates were blotted for sfGFP, indicating successful incorporation of HCK in the polypeptide chain.(B) Photoactivation of covalent reactivity in EGFP-Talin head-SpyCatcher003(K31TAG) in cell lysates.Lysates of HEK293T cells transfected with EGFP-Talin head-SpyCatcher003(K31HCK) and HCK RS with or without HCK and light activation were mixed with recombinant SpyTag003-Maltose-binding protein (SpyTag003-MBP), before Western blotting with anti-SpyCatcher003.(C) As in panel B, but analyzed by Western blot against EGFP.GFP variants are highly stable to proteasomal degradation, so GFP-linked fragments are commonly observed to accumulate in cells.54 Figure S2.Photostability of SpyCatcher003(K31HCK) in ambient light.Photostability of SpyCatcher003(K31HCK) in typical laboratory lighting.Lysates of HEK293T cells expressing EGFP-Talin head-SpyCatcher003(K31HCK), HCK tRNAs and HCK RS were mixed with 50 µM SpyTag003-MBP and incubated for the indicated time, either in a dimly lit cold-room (Low intensity, L) or by the laboratory window (High intensity, H).The positive control sample was activated with a UV transilluminator.Analysis by Western blot against EGFP.As in Fig.S1, GFP variants are highly stable to degradation by the proteasome, so GFP-linked fragments are observed to accumulate in cells.54

Figure S3 .Figure S4 .
Figure S3.Assessment of potential phototoxicity from 405 nm illumination.Talin knock-out cells were cultured in FluoroBrite DMEM with Trolox (orange bars) or without (blue bars) and exposed to 405 nm wide-field light for the indicated time at 45 mW/cm 2 , before resazurin-based cell viability assay.Viability is normalized to the No Light control, showing mean ± 1 SD for 3 replicates.Statistical comparisons against No Light control cells are shown for cells cultured with Trolox (black font) or no Trolox samples (gray font) using Oneway ANOVA and Dunnett's test: *** p<0.0001, ** p<0.001, ns p>0.05.